Abstract
Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg–Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln–His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872–878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr–Ser and Gln–Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix–loop–helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.
Highlights
Flavin-containing monooxygenases (FMOs) [1] catalyse the oxygenation of heteroatoms, such as nitrogen and sulfur, in various organic substrates, and have been studied both for their role in metabolism in higher eukaryotes, including humans [2,3], and for their contributions to microbial metabolism, in which they are able to catalyse the oxidation of amines [4] and amino acids such as ornithine [5]
A well-studied example of a bacterial FMO is that from Methylophaga aminisulfidivorans (Uniprot Q83XK4, mFMO), an enzyme of monomer molecular weight 46 kDa that was identified on the basis of its ability to form the pigment indigo through oxidative transformation of indole [8]. mFMO has been shown to catalyse the asymmetric sulfoxidation of a series of prochiral thioethers, when employed as part of a fusion enzyme with phosphite dehydrogenase for the recycling of the nicotinamide cofactor [9]
Studies of mFMO have shown a dependence for the phosphorylated cofactor NADPH, but the lower cost of the non-phosphorylated analogue, NADH, has meant that recent studies of FMOs have been directed towards enzymes that might employ that cofactor for flavin reduction
Summary
Flavin-containing monooxygenases (FMOs) [1] catalyse the oxygenation of heteroatoms, such as nitrogen and sulfur, in various organic substrates, and have been studied both for their role in metabolism in higher eukaryotes, including humans [2,3], and for their contributions to microbial metabolism, in which they are able to catalyse the oxidation of amines [4] and amino acids such as ornithine [5]. We recently reported the cloning, expression and structural characterisation of another FMO, named SMFMO, from the marine bacterium Stenotrophomonas maltophilia [10] This target was interesting as it displayed the ability to use either NADPH or NADH as the cofactor for reduction of the flavin. The structure of SMFMO was determined [10], and analysis of the nicotinamide cofactor binding loop revealed differences between NADPH-dependent mFMO [12,13,14] and SMFMO that might be significant in the recognition of the NADPH 2 ribose phosphate that distinguishes NADPH and NADH [10]. The resultant cell pellets were resuspended in 25 mL 50 mM Tris/HCl buffer pH 7.5, containing 300 mM sodium chloride (‘buffer’) per L of cell growth These suspensions were subjected to cell disruption using an ultrasonicator for 3 × 30 s periods at 4 ◦C with intervals of 1 min. Typical CFMO and PSFMO preparations yielded 20 mg and 7.5 mg pure protein per litre of cells, respectively
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